Dye staining of proteins to form non-covalent dye/protein complexes is a method widely employed in analytical biochemistry. It is used to detect proteins in electrophoretic procedures (Fazekas de St. Groth et al., Biochem. Biophys. Acta, 71, 377-391, 1963), to quantify proteins (Bradford, Anal. Biochem., 72, 248-254, 1976; Rinderknecht et al., Clin. Chem. Acta, 21, 197-203, 1968), and to detect and quantify analytes that bind to proteins (Scalbert, Plant Polyphenols, 259-280, 1992; Asquith and Butler, J. Chem. Ecology, 11, 1535-1544, 1985). A variety of dyes have been proposed and evaluated for usefulness in analytical procedures (Scalbert, 1992; Martin and Martin, Oecologia, 54, 205-211, 1982; Flores, Anal. Biochem., 88, 605-611, 1978; Datyner and Fennimore, Anal. Biochem., 55, 479-491, 1973; Atherton et al., Anal. Biochem., 232, 160-168, 1996; Sohl and Splittgerber, J. Chem. Education, 61(3), 262-264, 1991; Compton and Jones, Anal. Biochem., 151, 369-376, 1985). Two dyes with the most advantageous combination of protein specificity and sensitivity are Coomassie Brilliant Blue G-250 (CBB) (Bradford, 1976) and Remazol Brilliant Blue R (RBB) (Rinderknecht et al., 1968).
On the assumptions that (1) the detailed chemical structure of the CBB dye shown below ##STR1## is but one of several possible resonance structures and (2) the uncharged tertiary nitrogen is equivalent to that shown carrying the positive charge, Compton and Jones (1985) suggested that the first protonation occurs at one of two tertiary amines, followed by protonation at the other. The sulfonic acid groups are less susceptible to protonation than the amino groups, due to their strong acidities (the pKa of benzosulfonic acid is 0.70).
There is good evidence that the anionic blue CBB species binds to proteins (Atherton et al., 1996; Chial and Splittgerber, 1993; Compton and Jones, 1985). At the Bradford assay pH, the dye is mostly in a highly protonated reddish form with smaller proportions of green and blue forms. The green and blue species absorb light much more intensely than the red form at the 595 nm (Chial et al., 1993), the zero-protein assay mixture has a relatively low absorbance. The lower the assay pH, the greater the proportion of the red dye species and the lower the zero-protein absorbance at 595 nm. However, the assay pH must be at the same time reasonably high so that a small proportion of the blue form remains, since it is thought to be the protein-binding species of the dye.
Addition of protein to the dye reagent of low pH results in a marked decrease of the 470 nm absorption, a new shoulder at 595 nm, and little change at 650 nm. The three different absorbance maxima of the dye reagent-protein complex correspond to the three species of dye present: the doubly protonated, positively charged red species absorbing at 470 nm, the singly protonated neutral green species at 650 nm, and the deprotonated (anion) blue dye-protein complex at 595 nm. Based on the identical absorbance maxima of the dye-protein complex and the dye anion, it was concluded (Compton and Jones, 1985) that the bound dye species is in fact in the anion form. The equilibria shown above are forced to the right as the anion is bound by protein at pH 1.0 and lower.
However, Chial and Splittgerber (1993) showed that while the blue species of CBB dye is indeed the protein-binding form, the wavelength of its maximum absorbance is different from the wavelength maximum of the dye-protein complex. The extinction coefficients of the free blue dye species and the dye-protein complex also differ. The authors concluded that these factors could allow for detection of dye-protein binding at elevated pH where the free dye is present mostly in its blue anion form. They and others (Atherton et al., 1996; Congdon et al., 1993; Splittgerber and Sohl, 1989) have observed a red shift in absorbance maximum from 585 nm for the free blue species to 620 nm for the protein-bound dye species at pH 7.0.
CBB has advantages over other proteins-staining dyes because of its exceptionally high color intensity and position of its absorption peak (Fazekas de St. Groth et al, 1963). It is advantageous over RBB in terms of specificity for proteins (Compton and Jones, 1985), sensitivity, stability, speed, convenience and economy (Atherton et al., 1996; Sohl and Splittgerber, 1991). Proteins bound to CBB bind by hydrophobic interactions that are uniform for a wide variety of proteins, and the dye/protein complex has a higher extinction coefficient at .lambda.max than RBB dye/protein complexes. However, current methods for protein staining using CBB are disadvantageous because they denature proteins and, therefore, cannot be employed when it is important to preserve the protein in a native, functional form. Preserving the native form of stained proteins is important for some functional assays, like analyte binding or enzymatic activity. CBB methods of staining proteins are denaturing because they employ the dye in a cationic form, which typically requires the use of alcohol and acid solvents. These solvents, which tend to irreversibly denature proteins, are required to promote efficient solubilization of CBB due to its hydrophobic character, and because CBB is ordinarily used in its protonated form which has low solubility in aqueous, non-acidic and non-organic solvents. Unfortunately, there are no methods for non-covalently staining proteins with CBB that employ the dye in its deprotonated form that would be useful under non-denaturing conditions (i.e., in the absence of alcohol solvent(s) and at a pH greater than 4.0).
RBB staining methods have been employed that allow staining of some serum proteins, such as albumins, in a way that retains some functional activity of the proteins (Asquith and Butler, 1985). However, RBB has several disadvantages as a protein stain. RBB is even less soluble in aqueous media than CBB, and is a less stable stain that tends to "leak" from the protein into the solvent media, which lowers precision and reproducibility of measurements of the dye/protein complex. RBB has a lower extinction coefficient at .lambda.max than CBB and, therefore, lower sensitivity for detecting protein. It is not as specific for proteins, and stains primarily by ionic association with positively charged amino groups on proteins and is thus less uniform than CBB. RBB is highly negatively charged at pH levels above 5.0, which tends to cause aggregation and unwanted precipitation of proteins at higher pH levels. Further, the dye itself also tends to precipitate without protein in the presence of methanolic solvents, which may be required for the preparation of some analytes. Because of these problems, the use of RBB as a protein stain, and more particularly for maling dye/protein complexes useful in detecting analytes, is more limited than CBB.
The ability of some analytes to bind non-denatured proteins is the basis for certain methods of analyte detection, including, for example, the detection of tannins. Tannin detection by protein binding employs the distinguishing characteristic of tannins to bind and precipitate proteins (Giner-Chaves, Ph.D. Thesis, Cornell University, 1996; Hagerman and Butler, Methods in Enzymology, 234, 429-437, 1994; Hagerman, Phenolic Compounds in Foods and Their Effects on Health, 236-262, 1992; Haslam, et al., Id., 8-50; Scalbert, 1992; Field and Lettinga, Plant Polyphenols, 673-692, 1992; Dawra et al., Anal. Biochem., 132, 50-79, 1988). These methods have found wide application in biological and/or ecological studies for the detection of tannins in plant materials, feeds and foods (Hagerman, 1992; McArthur et al., Plant Defenses Against Mammalian Herbivory, Chap. 6, 103-114, 1991; Asquith and Butler, 1985; Mole and Waterman, Oecologia, 72, 137-147, 1987; Bate-Smith, Phytochemistry, 16, 1021-26, 1977, Hagerman and Butler, J. Agric. Food Chem., 28, 944-947, 1980; Swain, Herbivores, 657-682, 1979; Haslam and Lilley, Plant Flavanoids in Biology and Medicine, 53-65, 1986; Rinderknecht et al., 1968).
Methods for detecting tannins by protein precipitation differ in the choice of protein precipitated, as well as the parameters used for evaluating the precipitation. Three protein precipitation assays have been developed in this regard: (1) hemoglobin-precipitation, the "astringency assay" (Okuda et al., Chem. Pharm. Bull., 33, 1424-1433, 1985; Schultz et al., J. Agric. Food Chem., 29, 823-832, 1981; Bate-Smith, Phytochemistry, 12, 907, 1973; Phytochemistry 16, 1421, 1977); (2) .beta.-glucosidase-precipitation (Goldstein and Swain, Phytochemistry, 4, 185, 1965); and (3) Bovine Serum Albumin (BSA)-precipitation (Hagerman and Butler, J. Agric. Food Chem., 26(4), 809-812, 1978). Unfortunately, each of these assays has disadvantages. The hemoglobin-precipitation assay is inconvenient because it requires freshly drawn blood, and because some non-tannin phenols present in plant extracts absorb at the same .lambda.max wavelength (578 nm) used for measuring the amount of hemoglobin precipitated (Scalbert, 1992; Makkar et al., 1987; Martin and Martin, 1982). The .beta.-glucosidase assays are based on the ability of tannins to inhibit enzyme activity and rely on measurement of residual .beta.-glucosidase activity in a supernatant after protein precipitation in the presence of tannins (Martin and Martin, 1982; Hagerman and Butler, 1978). Such methods are cumbersome, time consuming and complicated.
The BSA-precipitation assay, originally introduced by Hagerman and Butler in 1978, is more useful because of speed and ease of use. However, the original method, and subsequent modifications thereof, have other drawbacks. As originally developed, the assay relies on direct measurement of tannins, rather than the protein in the tannin-protein complexes (Makkar et al., Anal. Biochem., 166, 435-439, 1987). Nonspecific binding of other phenols to tannin-protein complexes introduces a large error into the method (Hagerman and Butler, 1978). A modification of this assay measures proteins in a redissolved precipitate by the ninhydrin reaction which has limited interference from polyphenols (Mole and Waterman, 1987). This assay requires proteins in the precipitate to be hydrolyzed with 13.5N NaOH at 120.degree. C. before measurement of the released amino acids with ninhydrin (Makkar et al., 1987). This is technically more laborious and time consuming than direct measurement of a pre-labeled protein.
A modification that uses radioactively labeled BSA relies on measurement of the amount of radioactivity precipitated in the presence of tannins (Hagerman and Butler, 1980). Measurement of radioactive samples is expensive, requires special equipment, special expertise and trained staff. Therefore, many investigators prefer another modification that uses dye-labeled BSA (Asquith and Butler, 1985). This assay relies on covalently labeling the protein with the dye RBB, but suffers from all the disadvantages mentioned above for protein staining therewith. The disadvantages are such that an additional assay of protein determination (Lowry method) may be required to obtain reliable results. Furthermore, the assay is limited to measuring tannin-bound protein present in the precipitate and is unable to determine the amount of tannins remaining in the supernatant. This limits the accuracy of the assay and the range of tannin concentrations that can be determined because tannin precipitation of proteins strongly depends on the ratio of tannin to protein, thus some undetected tannins may remain present in the supernatant at certain tannin concentrations.
Accordingly, there is a need in the art for a simple, rapid, and economical method to make stable, dye/protein complexes using a sensitive and protein specific dye like CBB under conditions that do not irreversibly denature proteins. There is also a need to provide methods that are rapid, sensitive, reproducible, and economical for the detection of analytes, like tannins, that bind to dye/protein complexes and for an assay that allows accurate determination of tannin levels present both in a precipitate and supernatant fraction. The present invention fulfills these needs and provides other related advantages.